Name: in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons
Uploaded: 2010-10-08T17:00:00
Description: Watch this Scientific Journal Video about In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons at JoVE.com

The authors would like to thank Joseph LoTurco and Dennis Selkoe for helpful discussions on this technique. The authors thank the donors of the American Health Assistance Foundation, for support of this research.

The basic protocol for in utero electroporation has been described in detail in another JoVE article from the Kriegstein lab 1, 2. This technique was originally described in the Osumi lab 3 and our protocol is based upon one developed in the LoTurco lab 4. We will provide an overview of the our protocol for in utero electroporation of rat embryos, focusing on the most important details, followed by a description of our protocol that applies in utero electroporat...

<ol>
	<li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. , (2007).</li><li>Walantus, W., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E16+rat+embryos.">In utero intraventricular injection and electroporation of E16 rat embryos.</a> J Vis Exp. , (2007).</li><li>Takahashi, M., Sato, K., Nomura, T., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulating+gene+expressions+by+electroporation+in+the+developing+brain+of+mammalian+embryos.">Manipulating gene expressions by electroporation in the developing brain of mammalian embryos.</a> Differentiation. 70 (4-5), 155-1562 (2002).</li><li>Bai, J., Ramos, R. L., Ackman, J. B., Thomas, A. M., Lee, R. V., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+reveals+doublecortin+is+required+for+radial+migration+in+rat+neocortex.">RNAi reveals doublecortin is required for radial migration in rat neocortex.</a> Nat Neurosci. , 1277-1283 (2003).</li><li>Okada, A., Lansford, R., Weimann, J. M., Fraser, S. E., McConnell, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cells+in+the+developing+nervous+system+with+retrovirus+expressing+modified+green+fluorescent+protein.">Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.</a> Exp Neurol. 156 (2), 394-406 (1999).</li><li>Bai, J., Ramos, R. L., Paramasivam, M., Siddiqi, F., Ackman, J. B., LoTurco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+DCX+and+LIS1+in+migration+through+the+lateral+cortical+stream+of+developing+forebrain.">The role of DCX and LIS1 in migration through the lateral cortical stream of developing forebrain.</a> Dev Neurosci. , 144-156 (2008).</li><li>Molyneaux, B. J., Arlotta, P., Menezes, J. R., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtype+specification+in+the+cerebral+cortex.">Neuronal subtype specification in the cerebral cortex.</a> Nat Rev Neurosci. , 427-437 (2007).</li><li>Young-Pearse, T. L., Bai, J., Chang, R., Zheng, J. B., Loturco, J. J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Critical+Function+for+-Amyloid+Precursor+Protein+in+Neuronal+Migration+Revealed+by+In+Utero+RNA+Interference.">A Critical Function for -Amyloid Precursor Protein in Neuronal Migration Revealed by In Utero RNA Interference.</a> J Neurosci. 27, 14459-14469 (2007).</li><li>Young-Pearse, T. L., Chen, A. C., Chang, R., Marquez, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+APP+regulates+the+function+of+full-length+APP+in+neurite+outgrowth+through+interaction+with+integrin+beta1.">Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1.</a> Neural Develop. 3, 15-15 (2008).</li></ol>

In vitro study of primary neuronal cultures allow for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or misexpression constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero e...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cortical Neuron Preparation</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Media:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10X Hanks' Balanced Salt Solution (HBSS) (Ca+2 /Mg +2 free)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14185-052</td>
<td>&nbsp;</td>
<tr>
<td>10X Hanks' Balanced Salt Solution (HBSS) (with Ca+2 /Mg +2 )</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14065-056</td>
<td>&nbsp;</td>
<tr>
<td>1M HEPES pH 7.4</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15630-080</td>
<td>&nbsp;</td>
<tr>
<td>Dishes and Vials:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100 x 15 mm Petri Dishes</td>
<td>
</td>
<td>Fisherbrand</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>60 x 15 mm Petri Dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>351007</td>
<td>&nbsp;</td>
<tr>
<td>15 mL conical vial</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>62-547-205</td>
<td>&nbsp;</td>
<tr>
<td>50 mL conical vial</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>62-554-205</td>
<td>&nbsp;</td>
<tr>
<td>Dissection Tools:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scissors </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>91402-12</td>
<td>&nbsp;</td>
<tr>
<td>Standard Forceps</td>
<td>
</td>
<td>Fine Science Tools</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>Curved Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11273-20</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11255-20</td>
<td>&nbsp;</td>
<tr>
<td>Vannas spring scissors </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Miscellaneous:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25200</td>
<td>&nbsp;</td>
<tr>
<td>Reichert Bright-Line Hemacytometer</td>
<td>&nbsp;</td>
<td>Hausser Scientific</td>
<td>1490</td>
<td>&nbsp;</td>
<tr>
<td>Hand-Held Tally Counter</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>Z169021</td>
<td>&nbsp;</td>
<tr>
<td>Plating Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco's Modified Eagle Medium (D-MEM)</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960-051</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F4135</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Growth Medium:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NEUROBASAL Medium </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21103-049</td>
<td>&nbsp;</td>
<tr>
<td>B-27 Serum-Free Supplement </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>17504-044</td>
<td>&nbsp;</td>
<tr>
<td>GlutaMAX -I Supplement</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>35050-061</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicin Reagent Solution </td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>15750-060</td>
<td>&nbsp;</td>
<tr>
<td>Immunostaining:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fixative, Washes, and Blocking Buffer:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P6148</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T9284</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Serum</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>017-000-121 </td>
<td>&nbsp;</td>
<tr>
<td>Antibodies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>beta-III tubulin antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>MAB1637</td>
<td>&nbsp;</td>
<tr>
<td>MAP2 antibody</td>
<td>&nbsp;</td>
<td>Chemicon</td>
<td>AB15452</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy3 anti-mouse</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>715-166-151</td>
<td>&nbsp;</td>
<tr>
<td>Donkey Cy2 anti-chicken</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>703-226-155 </td>
<td>&nbsp;</td>
<tr>
<td>DAPI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>D3571</td>
<td>&nbsp;</td>
<tr>
<td>Slide Preparation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CC2 Coated Two-Chamber Slides </td>
<td>&nbsp;</td>
<td>Lab-Tek</td>
<td>154852</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent Mounting Media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>24 x 60 mm Micro Cover Glasses</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>48393-106 </td>
<td>&nbsp;</td>
<tr>
<td>Clear nail polish</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72180</td>
<td>&nbsp;</td>
<tr>
<td>Electroporation:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>995-2949</td>
<td>&nbsp;</td>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>4015809TV</td>
<td>&nbsp;</td>
<tr>
<td>buprenorphine</td>
<td>&nbsp;</td>
<td>Henry Schein</td>
<td>1118217</td>
<td>&nbsp;</td>
<tr>
<td>Picospritzer III</td>
<td>&nbsp;</td>
<td>Parker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BTX square wave electroporator</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BTXECM830</td>
<td>&nbsp;</td>
<tr>
<td>Tweezertrodes, 7 mm, platinum</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>450488</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

Watch this Scientific Journal Video about In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons at JoVE.com

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

Research

JoVE Journal

Neuroscience

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Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

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High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

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Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

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The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...